Effects Of APOBEC Mutagenesis On Immune Infiltration,Immunotherapy Response And Its Mechanisms In Esophageal Squamous Cell Carcinoma

Background and objective:Somatic mutations accumulate throughout the process of tumor evolution.The cancer genome exhibits certain patterns of mutation,known as mutational signatures,reflecting the footprints of exogenous and endogenous mutational processes.The apolipoprotein B mRNA editing enzyme catalytic polypeptide(APOBEC)-related signatures,SBS2 and SBS13,are detected in at least 22 different cancer types and reported to be near-universally exhibited in esophageal squamous cell carcinoma(ESCC),indicating that APOBEC mutagenesis is an essential and mandatory process due to its prevalence in ESCC.Moreover,it is reported that SBS2 and SBS13 present in majority of ESCC samples and account for about 25%of the mutation burden,revealing that APOBEC mutagenesis is a crucial step during the evolutionary history in ESCC.The APOBEC signatures are characterized by C-to-T or C-to-G changes at TCW motifs(where W refers to A or T).APOBEC3 subfamily members,which catalyze the deamination of cytosine to uracil,are responsible for the vital mutational process but which one predominantly contribute to APOBEC mutagenesis remains unknown in ESCC.Immunotherapy has become a promising strategy for ESCC,and the characteristics of the tumor immune microenvironment are prerequisites for patient stratification.The biological functions of APOBEC mutagenesis are diverse in different types of cancer,but its functional roles and mechanisms in tumor immunogenicity and immunotherapy response have not been fully understood in ESCC.Methods:In the present study,we collect matched multi-omics data of 169 patients with ESCC from three previously published cohorts.To dissect the effects of APOBEC mutagenesis on tumor immune infiltration,we perform mutation enrichment analysis based on the somatic mutation data and employ CIBERSORT,ESTIMATE and TMEscore to conduct tumor microenvironment deconvolution using bulk RNA sequencing(RNA-seq)data,which is verified using single-cell RNA sequencing(scRNA-seq)data and multiplex immunofluorescence.What’s more,we identify which member of APOBEC3 subfamily is the dominant mutator through the 20 bp upstream and downstream base sequence of the mutated base and the expression of the APOBEC3 subfamily members,and illustrate the underlying molecular mechanisms of immune activation for APOBEC mutagenesis in ESCC by conducting a set of functional assays.Results:We find that APOBEC mutagenesis prolongs overall survival(OS)of ESCC patients.The reason for this outcome is probably due to high anti-tumor immune infiltration,immune checkpoints expression and immune related pathway enrichment,such as interferon(IFN)signaling,innate and adaptive immune system.The elevated AOBEC3A(A3A)activity paramountly contributes to the footprints of APOBEC mutagenesis.Mechanistically,upregulated A3A exacerbates cytosolic double-stranded DNA(dsDNA)accumulation,thus stimulating cGAS-STING pathway.Simultaneously,A3A is associated with immunotherapy response which is predicted by Tumor Immune Dysfunction and Exclusion(TIDE)algorithm and validated in a clinical cohort.These findings systematically elucidate the clinical relevance,immunological characteristics,prognostic value for immunotherapy and underlying mechanisms of APOBEC mutagenesis in ESCC,which demonstrate great potential in clinical utility to facilitate clinical decisions.Conclusion:In conclusion,we illustrated whether and how APOBEC mutagenesis activate immune response using whole genome/exome sequencing(WGS/WES),bulk RNA-seq,scRNA-seq data and functional assays.These findings uncovered a new biomarker to accelerate the clinical application and improve immunotherapy effects in ESCC.